PRINCIPAL INVESTIGATOFVPROGRAM DIRECTOR: 
Sandra Handwerger, M.D. 
peptidoglycan precursors) in the presence of 200 ug/ml of 
vancomycin. There was no effect on growth of VR as monitored by 
optical density when concentrations of 0.5 x MIC or greater of 
each of the 3 antibiotics was tested. However, sub-MIC 
concentrations of cycloserine or bacitracin in combination with 
vancomycin yielded growth inhibition of the susceptible strain 
688. Results of a typical experiment are shown in figure 1 
(appendix A) . Although they do not exclude the possibility, 
these findings suggested that hyperproduction of peptidoglycan 
precursors might be an unlikely mechanism of antibiotic 
resistance . 
To further explore this hypothesis, UDP-linked 
peptidoglycan precursors of strains VR, VR1, and 688 were 
assayed (15,44). One liter cultures 7 of each stgain were grown in 
MRS medium to mid log phase (5 x 10 to 2 x 10° cells per ml) , 
chilled rapidly, and harvested by centrifugation. Cells were 
boiled and then extracted with cold trichloroacetic acid (TCA) , 
final concentration 5%, for 20 minutes. Cell debris was removed 
by centrifugation (15,000 x g for 20 minutes). The supernatant 
fluid was extracted three times with ether to remove TCA, and 
the pH adjusted to 7.0. Samples were then dried, resuspended in 
1 ml water, applied to a Sephadex G25 column (16 x 100 cm) , and 
eluted with water. Two ml factions were collected. Fractions 
were assayed for N-acetyl hexosamines by the method of Ghuysen 
(17) after mild acid hydrolysis (0.1 N HCl at 100°C for 5 
minutes) . * 
Figure 2 (appendix A) ^shows the results of a typical 
experiment, using 5.6 x 10 1 cells of strain VR1 . N-acetyl 
hexosamine containing compounds were eluted in two peaks, as 
described by Mengin-Lecreulx et al (44) , the first corresponding 
to UDP-' -acetyl-muramic acid and its peptide derivatives, the 
second t3 UDP-N-acetyl-glucosamine . No significant difference 
was apparent in the concentrations of total UDP-hexosamines 
between the two resistant strains VR and VR1 and the susceptible 
strain 688 (range 30 to 42 nmol per 10 1 cells) , suggesting that 
significant hyperproduction of cell wall precursors is an 
unlikely mechanism of resistance. However, this preliminary 
separation does not distinguish among individual precursors; 
alterations in the structure or relative proportions of 
precursors have not been excluded . Therefore , the pooled 
fractions will be subjected to high pressure liquid 
chromatography for more detailed analysis (see below) . 
c) Change in the composition of cell wall peptides ; Given 
the specificity of vancomycin for D-alanyl-D-alanine the 
possibility was tested that vancomycin resistance in strain VR 
is related to a major change in cell wall composition, e.g. 
replacement of D-alanine with another amino acid. Purified cell 
walls were prepared as follows: Cultures of VR were grown to 
mid log phase in c+y medium, chilled rapidly, and harvested by 
centrifugation. The cells were suspended in chilled saline 
phosphate buffer and boiled in sodium dodecyl sulfate (SDS, 
final concentration 4%) for 30 minutes. After extensive washing, 
acid washed glass beads were added and the cells broken with 10 
one-minute pulses on a vortex mixer. Unbroken cells were removed 
by centrifugation. The cell wall fraction was harvested by 
Recombinant DNA Research, Volume 13 
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